Direct Fluorescent Antibody Staining Research Articles

Diagnosis Of Chlamydia Trachomatis Urethral Infection In Symptomatic And Asymptomatic Men By Testing First-Void Urine In A Ligase Chain Reaction Assay

A multicenter study compared ligase chain reaction (LCR) of Chlamydia trachomatis plasmid DNA with culture of urethral swab specimens from 542 men (study A); a second study (B) compared LCR of first-void urine (FVU) with urethral swab cultures from 1043 men. Discordant results were resolved with direct fluorescent antibody staining of sediments from the FVU or urethral culture specimen and with a second LCR directed against a fragment of the major outer membrane protein gene. Test performance was calculated on the basis of an expanded reference standard. The LCR plasmid assay had a sensitivity of 98.0% in study A and 93.5% in study B; specificity was 99.8%-100%. The sensitivity of culturing urethral swabs from all study sites was 68.2% (range by sites, 40.0%-84.6%). The presence or absence of urethral symptoms did not alter the results. Use of this LCR test should allow more meaningful investigation and treatment of C. trachomatis infections in men.

Role of confirmatory PCRs in determining performance of Chlamydia Amplicor PCR with endocervical specimens from women with a low prevalence of infection

The role of confirmatory PCR assays for determining the performance of Chlamydia Amplicor PCR for endocervical specimens from women with a low prevalence of infection was evaluated. An endocervical swab was collected from 770 women and tested by culture or direct fluorescent antibody (DFA) staining. A second swab was tested by Chlamydia Amplicor PCR (Roche Molecular Systems, Branchburg, N.J.). Discordant results were resolved by three confirmatory PCRs: one targeting the plasmid by using different primers and two directed to the major outer membrane protein (MOMP) gene. Of the 30 swabs that were positive by culture or DFA (3.9%), 27 were positive by Amplicor PCR. An additional five swabs were positive by Amplicor PCR but negative by culture or DFA. Both plasmid and MOMP confirmatory PCRs identified the five culture-DFA negatives and the three Amplicor negatives as true positives. The three specimens originally classified as negative by Amplicor PCR were positive on repeat Amplicor testing. After resolution of the discordant results by confirmatory PCR testing, the sensitivity of the initial Amplicor PCR was 91.4% (32 of 35 specimens), changing to 100% after storage and repeat testing. The specificity of Amplicor PCR was 100% (735 of 735 specimens). Our results demonstrated that plasmid and MOMP confirmatory PCRs worked equally well in resolving false-positive and false-negative Amplicor PCR results. Some specimens may contain inhibitors of Amplicor PCR which may disappear with time.

Pneumonia in Military Recruits

Lower respiratory disease is a major source of morbidity in military recruits, with hospitalization rates for pneumonia more than 30 times that of the non-recruit population. The etiologic agent remains unknown in over 75% of cases. This study prospectively examined the etiology of pneumonia among recruits at Naval Training Center, San Diego, California. Recruits presenting with cough, fever, or shortness of breath and pulmonary infiltrates on chest X-ray were eligible for enrollment. A standardized scoring form and focused physical exam were completed on each subject. Sputum specimens were obtained for Gram's stain and culture, DNA probing for Legionella and Mycoplasma species, and direct fluorescent antibody staining for Legionella. Acute and convalescent serologies were performed for adenovirus, influenza A and B, Mycoplasma pneumoniae, Chlamydia group, and respiratory syncytial virus. Of 110 eligible patients, 100 consented to enrollment and 75 patients completed the study. Etiologic diagnoses were obtained in 40 of the patients (53%). M. pneumoniae, Haemophilus influenzae, and viruses accounted for the majority of infections. Mixed infections were seen in six patients. Forty-seven percent of patients had no diagnosis established. Pneumonia in this series of military recruits was frequently caused by M. pneumoniae and H. influenzae. Fifty percent of cases were undiagnosed with routinely available laboratory methods. Further studies are warranted to more clearly define the etiologic agents of recruit pneumonia and the utility of prophylactic measures.

Prediction of cytomegalovirus pneumonia after marrow transplantation from cellular characteristics and cytomegalovirus culture of bronchoalveolar lavage fluid.

Bronchoalveolar lavage (BAL) was performed between days 35 and 45 posttransplant in 37 marrow recipients without clinical or radiologic evidence of cytomegalovirus (CMV) pneumonia to determine if the presence of CMV or cellular characteristics was predictive of subsequent development of CMV pneumonia. Analysis of BAL fluid included culture and direct fluorescent antibody staining for CMV, total mononuclear cell count, lymphocyte count, and lymphocyte subsets. Patients were followed to day 110 after transplant for development of CMV pneumonia. Weekly cultures of blood, urine, and throat were also obtained. BAL was positive for CMV in 7 patients, of whom 3 developed CMV pneumonia. It was negative for CMV in 30 patients, of whom 7 developed CMV pneumonia. The positive and negative predictive values for detection of CMV in day 35-45 BAL were 43% and 78%, for CMV viremia 66% and 87%, and for detection of CMV in urine and throat 41% and 94%, respectively, for subsequent CMV pneumonia. In 9/10 patients who subsequently developed CMV pneumonia, excretion of CMV from BAL, blood, urine or throat preceded CMV pneumonia, and BAL was the earliest site of CMV excretion in all nine. Only one patient did not excrete before developing CMV pneumonia. In a stepwise logistic regression of risk factors for developing CMV pneumonia, only CMV viremia was significant (P = 0.002) although the total BAL mononuclear cell count approached significance (P = 0.053). The percentage of lymphocytes in BAL and CMV excretion in BAL, urine, and throat were not significant. The positive predictive value of BAL for subsequent CMV pneumonia was no greater than that of culture of urine and throat and less than that of viremia in our patients.

Limited value of two widely used enzyme immunoassays for detection ofChlamydia trachomatis in women

Enzyme immunoassays (EIAs) are widely used to diagnose chlamydial infections in patients attending genitourinary medicine clinics. They are relatively easy to perform and are suitable for testing large numbers of samples. The objective of this study was to determine what proportion of women with chlamydial infection, defined as the presence of Chlamydia trachomatis in a cervical smear or deposit and/or in the urinary tract, detected by means of a sensitive direct fluorescent antibody test could also be identified by using two commercially available EIAs to test cervical samples. On hundred fifty-one women attending the genitourinary medicine clinic at St. Mary's Hospital, London, were enrolled. The use of the Chlamydiazyme (Abbott Diagnostics, UK) and MicroTrak (Syva, UK) EIAs resulted in the identification of only 56% and 63%, respectively, of women with chlamydial infection detected by direct fluorescent antibody staining. Thus, the EIAs available for detection of chlamydiae in cervical samples are inadequate for identifying all infected women. Improvement might be achieved by testing multiple samples or by resorting to tests of greater sensitivity.

Identification of influenza A virus by shell vial culture and two commercially available antigen detection methods

Background: Effective use of amantidine and rimantidine for treating patients and for reducing transmission requires rapid diagnosis of influenza A. Rapid culture methods require 1–2 days to detect influenza A virus. Direct fluorescent antibody (DFA) staining and enzyme immunoassay (EIA) can detect influenza A antigen within 1–4 h. Objectives: We compared DFA staining using the Bartels viral respiratory panel and the Directigen FLU-A EIA with shell vial centrifugation culture. Study design: Ninety-seven fresh specimens from a variety of respiratory sources and transported from hospitals throughout the USA to our national referral laboratory were tested. A true positive was defined as culture positive or both antigen tests positive. Results: Fifteen specimens were true positive. Sensitivity with culture was 93%, EIA 67%, and DFA 47%. Specificity was excellent with all three methods: 100%, 98%, 99%. Culture detected additional viruses that can cause respiratory tract disease: herpes simplex, cytomegalovirus, respiratory syncytial, influenza B, and adenovirus. Fourteen (70%) of 20 frozen specimens previously positive for influenza A were positive on retest by EIA. Overall sensitivity of EIA compared with culture using 35 positive specimens was 69%. Conclusions: These results suggest that the rapid EIA is useful to screen for influenza A, but that critical antigen-negative specimens should be submitted to a virology laboratory for culture for optimal sensitivity and for recovery of other viruses.

Cholera DFA: An improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1

An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC). An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well asimproved photographic recording of results without loss of fluorescence intensity. When tested for specificity, all 30 strains of V. cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study. A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods. Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V. cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens. The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

Identification of influenza A virus by shell vial culture and two commercially available antigen detection methods

Background: Influenza continues to be a major cause of morbidity and mortality especially in the elderly and persons with underlying disease. Shell vial cell culture and antigen detection techniques may speed up diagnosis and enable better patient treatment and management. Objectives: To compare shell vial centrifugation culture with commercially available direct fluorescence and enzyme immunoassay kits using a variety of respiratory specimens. Study design: To detect influenza A virus, we compared direct fluorescent antibody (DFA) staining using the Bartels Viral Respiratory Panel and the Directigen FLU-A enzyme immunoassay (EIA) with shell vial centrifugation culture. Ninety-seven fresh specimens from a variety of respiratory sources, and transported from hospitals throughout the U.S. to our national referral laboratory, were tested. Results: Fifteen specimens were true positives: culture positive or both antigen tests positive. Sensitivity with culture was 93%, EIA 67%, and DFA 47%. Specificity was excellent with all three methods: 100%, 98%, 99%. Culture detected additional viruses that can cause respiratory tract disease: herpes simplex, cytomegalovirus, respiratory syncytial, influenza B, and adenovirus. Fourteen (70%) of 20 frozen specimens previously positive for influenza A were positive on retest by EIA. Overall sensitivity of EIA compared with culture using 35 positive specimens was 69%. Conclusions: The rapid EIA is useful to screen for influenza A, but critical antigen-negative specimens should be submitted to a virology laboratory for culture. Shell vial cultures can provide a sensitive and universal diagnostic system for influenza A and a variety of other viruses.

Evaluation of the Amplicor Chlamydia trachomatis test versus culture in genital samples in various prevalence populations.

To evaluate a newly developed polymerase chain reaction (PCR) assay, Amplicor C trachomatis for the detection of C trachomatis in genital samples using cell culture for comparison. 501 patients (431 women and 70 men) attending an STD clinic in Hôpital Pellegrin (high-risk population) and gynaecological clinics (low-risk population) in Bordeaux, France. The genital samples (cervical and urethral) were tested for the presence of C trachomatis using the Amplicor test and using standard cell culture identified by the immunofluorescence test using a monoclonal antibody to C trachomatis. Discrepancies between the results of culture and Amplicor were further analysed by major outer membrane protein gene (omp1)-PCR of the specimens taken in transport media and by direct fluorescent antibody (DFA) staining of elementary bodies in culture transport tubes. After analysis of discrepancies, the revised sensitivity and specificity of PCR were 95.3% and 100% and the positive and negative predictive values were 100% and 99.5%, respectively. The present results indicate that the Amplicor assay is rapid, specific and more sensitive than the culture method. This test provides an excellent non-culture method for the detection of C trachomatis in various prevalence populations.

Diagnostic utility of PCR-enzyme immunoassay, culture, and serology for detection of Chlamydia pneumoniae in symptomatic and asymptomatic patients

To assess the utility of PCR-enzyme immunoassay (EIA) for diagnosis of acute infection with Chlamydia pneumoniae, we compared tissue culture, PCR-EIA, direct fluorescent-antibody (DFA) stain, and serology in studies with 56 patients with respiratory symptoms and 80 asymptomatic persons. Thirty-five patients were positive by either culture or PCR-EIA, and 101 were negative by both assays. Thirty specimens from symptomatic patients and one from an asymptomatic patient were culture positive; 23 of these were also PCR-EIA positive. Of the eight culture-positive, PCR-EIA-negative specimens, five were DFA negative and three were DFA positive. Four additional specimens were culture negative and PCR-EIA positive; of these, three were DFA positive and one was DFA negative. When we used culture- and/or DFA-positive results as a reference or "gold standard," the sensitivity and specificity of PCR were 76.5 and 99.0%, respectively. When we used PCR- and/or DFA-positive results as the reference, the sensitivity of culture was 87.5%. On the basis of single acute serum specimens, only 8 of these 35 patients had diagnostic antibody titers. Of the asymptomatic patients, 75% had immunoglobulin G or immunoglobulin M antibody to C. pneumoniae; 15 (18.8%) of these had antibody levels considered to be diagnostic of acute infection. This multicenter study indicates that culture and/or PCR-EIA is more reliable for prompt diagnosis of C. pneumoniae infection than single-point serology alone.

Evaluation of an enzyme immunoassay for detection of Chlamydia trachomatis in urine of asymptomatic men.

In a study of 1,486 men attending two sexually transmitted disease clinics, of whom 891 had no symptoms of urethritis, we compared an enzyme immunoassay (EIA) (Baxter-Bartels, formerly Northumbria AntigEnz) of urine sediment to urethral culture for the detection of Chlamydia trachomatis. C. trachomatis prevalence by culture alone was 7.7% in asymptomatic men and 10.9% in symptomatic men. Discrepant results between EIA of urine and urethral culture were evaluated by direct fluorescent-antibody staining (DFA) for elementary bodies in urine sediment or in culture transport media. When chlamydial infection was defined as either a positive urethral culture or positive EIA confirmed by DFA, chlamydia prevalence increased to 8.9% in asymptomatic men and 11.6% in symptomatic men. The urine EIA sensitivity, specificity, and positive and negative predictive values for chlamydia detection in asymptomatic men were 84.8, 99.3, 91.8, and 98.5%, respectively, with nearly identical results for symptomatic men. The sensitivities of urethral culture alone compared with the combination of urethral culture and urine EIA (with DFA confirmation) were 87.3 and 94.3% for asymptomatic and symptomatic men, respectively. The present EIA of urine sediment is both highly sensitive and specific for the detection of C. trachomatis in asymptomatic men, thus providing a noninvasive screening method for chlamydia infection in asymptomatic men attending sexually transmitted disease clinics.

Detection of Legionella species in sewage and ocean water by polymerase chain reaction, direct fluorescent-antibody, and plate culture methods.

Legionella spp. are ubiquitous in most environmental water sources; however, sewage treatment plants have not been examined as potential environmental reservoirs for these bacteria. This study used polymerase chain reaction, direct fluorescent-antibody staining, and culture methods to examine raw and treated sewage, ocean-receiving waters, and nearshore coastal environments for the presence of Legionella pneumophila and other Legionella spp. The study concluded that Legionella spp. are present in all phases of sewage treatment and that population numbers do not significantly decline through the treatment process. Ocean-receiving waters located 5 miles offshore, where the treated sewage is discharged, were found to contain Legionella spp., but ocean water between the discharge site and coastal bathing beaches was negative. This suggests that the Legionella spp. from the ocean discharge site were not reaching the nearshore beach waters. A flood control channel and river that entered the ocean were found to contain Legionella spp., and a nearby beach swimming area was also found to be positive, suggesting that land runoff from the flood control channel and river were the source of the Legionella spp. in the beach water samples that tested positive.

Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens

A clinical evaluation of the Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis in endocervical swabs (Roche Molecular Systems, Branchburg, N.J.) is described. This new clinical system used one-step sample preparation, amplification with biotinylated cryptic plasmid primer pairs (CP24-CP27), uracil-N-glycosylase (AmpErase), and a microtiter format for amplicon capture and detection. Culture with McCoy cells in duplicate 1-dram (3.697-ml) vials with fluorescent immunostaining was the reference system. Endocervical swab samples from 945 women provided 74 culture-positive specimens, of which PCR detected 71. The initial PCR result was positive for 12 additional specimens. Arbitration of the PCR-positive, culture-negative samples by PCR with major outer membrane protein primers, duplicate culture, elementary body direct fluorescent-antibody staining, and DNA extraction PCR showed that all 12 samples were positive for chlamydia, raising the number of truly positive samples from 74 to 86. After arbitration the true sensitivities of PCR and culture were 96.5 and 86%, respectively (P = 0.02). Specificities for both were 100%. For PCR, the positive and negative predictive values were 100 and 99.7%, respectively. Total test efficiency was 99.7%. A high-test-volume (121 samples) timing study with all items included in the College of American Pathologists work load method indicated that this PCR format took approximately 3 min per sample. Because of the high sensitivity, specificity, and improved ease of handling, we found PCR to be a good alternative to culture for detection of C. trachomatis.

Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice

In a modification of the direct epifluorescent filter technique (DEFT), direct fluorescent antibody staining was used for the rapid (<1 h), specific enumeration of foodbome Escherichia coli 0157:H7 by epifluorescence microscopy. Cell counts obtained by this method (Ab-DEFT) correlated well with DEFT counts obtained by acridine orange staining and with viable plate counts ranging from 108 to 101 cells per ml for pure cultures in buffer. Ab-DEFT also was effective for counting E. coli 0157:H7 cells inoculated into milk and juice; the sensitivity limit was about 103 for milk. The highly specific nature of the technique was demonstrated by enumeration of E. coli 0157:H7 cells in the presence of large numbers of indigenous spoilage microorganisms in milk. This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step.

Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay

Screening for Chlamydia trachomatis infection in men has traditionally been limited to men who present with urethral symptoms, thereby limiting the detection of asymptomatic chlamydia infection in men. In order to effectively screen both symptomatic and asymptomatic men, we evaluated a newly developed polymerase chain reaction (PCR) assay, Amplicor C. trachomatis, from Roche Molecular Systems for the detection of C. trachomatis in urine specimens in comparison with urethral culture. A total of 530 male urine specimens were collected from 322 symptomatic and 208 asymptomatic men attending two sexually transmitted disease clinics in Baltimore, Md. The prevalence of C. trachomatis by culture was 9.8% (10.6% in symptomatic men and 8.2% in asymptomatic men). Compared with culture, the sensitivity of the PCR was 92.8%, the specificity was 94.7%, the positive predictive value was 68.4%, and the negative predictive value was 99.1%. Discrepant results between culture and PCR were further analyzed by direct fluorescent-antibody staining of elementary bodies in urine sediment and in culture transport vials and by major outer membrane protein PCR of transport media for specimens with negative culture. The revised sensitivity and specificity of PCR for urine were 95.0 and 99.8%, respectively, and the positive and negative predictive values were 98.7 and 99.1%, respectively. The sensitivity of culture compared with PCR and/or direct fluorescent-antibody staining was 68.4%. These results indicate that the PCR assay is a highly sensitive and specific assay for the detection of C. trachomatis in male urine specimens and provides a noninvasive technique for routine screening of chlamydia infection in both symptomatic and asymptomatic men.

Pertussis in Missouri: evaluation of nasopharyngeal culture, direct fluorescent antibody testing, and clinical case definitions in the diagnosis of pertussis.

No diagnostic test for pertussis in routine use in the United States has both high sensitivity and high specificity. During a statewide increase in the incidence of pertussis in Missouri, we studied the clinical features of 153 patients with suspected pertussis in the Greater St. Louis area from whom a specimen for pertussis culture had been taken between 15 May and 19 September 1989. In this cross-sectional study, nasopharyngeal cultures were more likely to be positive for persons whose specimens were collected < 21 days after cough onset (adjusted rate ratio [RRa] and 95% confidence interval = 3.4; 1.5-8.0) and who were not receiving erythromycin/sulfamethoxazole prior to the culture [RRa = 5.8; 0.8-40.6], who had received fewer than three prior doses of pertussis vaccine [RRa = 1.8; 0.8-4.2], and whose specimen was in transit to the laboratory for < 4 days [RRa = 2.0; 0.8-5.5]. Among children < 5 years of age, spasmodic cough plus a lymphocytosis of > 10,000/mm3 was the acute symptom complex associated with the highest predictive value for a positive culture result (67%). Cough for > or = 14 days plus whoop was sensitive (81%) and specific (58%) for identifying children with culture-confirmed pertussis. Direct fluorescent antibody staining performed well as a screening test for pertussis but requires substantial commitment of personnel and resources. In the absence of a positive culture result, clinical case definitions should be used for decision making (e.g., initiation of antimicrobial therapy and routine case reporting).

Sequential Culturing Method Improves Recovery of Legionella spp. from Contaminated Environmental Samples

Investigations were undertaken to improve detection and isolation of Legionella spp. from samples containing a large number of non-legionellae isolates. The direct fluorescent antibody staining technique was used in conjunction with a sequential culturing method which was developed to improve the recovery rate of Legionella spp. from such samples. The technique for enrichment and isolation of Legionella spp. from environmental samples includes storage at 4 degrees C and repeated culture on freshly prepared media. Heat and acid treatments were included when deemed appropriate. A DNA probe was used for confirmation of Legionella. Treatment of the water samples, as described, and co-cultivation with amoebae naturally present in the samples are concluded to be responsible for increased success in recovery of Legionella isolates.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

False-positive Chlamydiazyme results during urine sediment analysis due to bacterial urinary tract infections

Our study examined whether urinary tract infections (UTIs) would cause false-positive results when urine sediment was tested with the Chlamydiazyme (CZ) system. Thirty-six infected urine samples and fifteen controls were studied. All controls were negative. Forty-seven percent of Escherichia coli UTIs (n = 30) and 100% of Klebsiella pneumoniae UTIs (n = 4) were positive on CZ testing of urine sediment. Nine E. coli UTIs positive by CZ were negative by direct fluorescent-antibody staining. When suspensions of the pure cultures were analyzed, 47% of E. coli and 100% of K. pneumoniae samples were CZ positive. False-positive results were not related to organism biotype or urine characteristics, including pH, specific gravity, and leukocyte count. We conclude that the presence of a UTI and also bacterial contamination must be ruled out prior to urine sediment testing.

Isolation of Chlamydia trachomatis from amniotic fluid

A 26-year-old white woman had premature rupture of membranes at 35 weeks' gestation. Cervical specimens initially demonstrated group B streptococci and Chlamydia trachomatis. Amniocentesis was performed and the amniotic fluid was positive for chlamydia by direct fluorescent antibody stain. The Gram stain was negative. The patient had an elevated white blood cell count. Labor was induced because of suspected chorioamnionitis. A 2120-g female infant was delivered with an Apgar score of 9 at 1 and 5 minutes. The infant's eye and nasopharyngeal specimens were positive for chlamydia by direct fluorescent antibody stain. After delivery, both Ureaplasma urealyticum and C trachomatis were isolated from the amniotic fluid and fetal membranes. This is the first reported case of chlamydial isolation in amniotic fluid.